(1) Field of the Invention
The present invention relates to a method for assaying peroxidase enzyme activity and/or the ability of a compound (or mixture) to serve as a reducing substrate in a peroxidase catalyzed reaction (i.e. its suitability or activity for this purpose). In particular the present invention uses a hydroperoxyphenyl alkene as a reactant in the assay which is reduced to the corresponding hydroxide. This work was sponsored by the National Institute of Health (GM23642).
(2) Prior Art
Peroxidases reduce hydroperoxides to alcohols at the expense of electron donors. (FIG. 6). Theoretically, peroxidase activity can be assayed by measuring either the reduction of hydroperoxide or the oxidation of an electron donor (Saunders, B. C., Helmes-Siedle, A. C. and Stork, B. P. (1964) in Peroxidase, Butterworth, Washington). Residual hydroperoxide can be determined titrimetrically or spectrophotometrically but the procedures are time-consuming and of limited specificity (Saunders, B. C., Helmes-Siedle, A. C. and Stork, B. P. (1964) in Peroxidase, Butterworth, Washington; Putter, J. (1962) Hoppe Seylers Z. Physiol. Chem. 329, 40-51; Mair, R. D., Hall, R. T. (1971) in Treatise on Analytical Chemistry, Part II, Vol. 14-(Kolthoff, I. M., Elving, P. J. eds.) Wiley-Interscience New York, pp. 295-434). Production of alcohol has been quantitated for cumene hydroperoxide and fatty acid hydroperoxides but these compounds are not substrates for many heme-containing peroxidases. Fatty acid hydroperoxides undergo rearrangements to non-ultraviolet absorbing compounds in the presence of heme complexes. Therefore, assays based upon their formation are not general. Most quantitative assays for peroxidase activity are based on the conversion of an electron donor into a chromogenic, fluorometric, or chemiluminescent species. A broad range of electron donors are oxidized by heme-peroxidases (Chance, B. and Maehly, A. C. (1964) in Methods in Enzymology (Colowick, S. D. and Kaplan, N. O. eds) Vol. 2, p. 764-775, Academic Press, New York). Frequently used electron donors are guaiacol (Chance, B. and Maehly, A. C. (1964) in Methods in Enzymology (Colowick, S. D. and Kaplan, N. O eds) Vol. 2, p. 764-775, Academic Press, New York; Nagataki, S., Uchimura, H. Masuyama, Y. and Nakao, K. (1973) Endocrinology 92, 363-371; Nagusaka, A. and Hidaka, H. (1976) J. Clin. Endocrinol. Metab. 43, 152-158; Hosoya, T., Kondo, Y, and Ui, N. (1962) J. Biochem. 52, 180-189; Horuchi, T. and Yoshizaki, T. (1982) Anal. Biochem. 126, 276-284; Maehly, A. C. and Chance, B. (1954) in Methods of Biochemical Analyses (Glick, D. ed.) Vol. I, pp. 357-424, Interscience, New York), mesidine (Saunders), various leuko-dyes (Saunders; Nickel, K. S. and Cunningham, B. A. (1969) Anal. Biochem. 27, 292-299), luminol (Puget, K., Michelson, A. M. and Arameus, S. (1977) Anal. Biochem. 79, 447-456), phenols (Zaitsu, K. and Ohkura, Y. (1980) Anal. Biochem. 109, 109-113; Makinen, K. K. and Tenovuo, J. (1982) Anal. Biochem. 126, 100-108) and reduced cytochrome c (Saunders; Puffler). Many of these compounds produce multiple products (Saunders, Chance) or undergo reactions with time courses markedly delayed relative to peroxide reduction (Saunders). Therefore, the stoichiometry of peroxide reduction cannot always be determined. In addition, significant electron donor specificity exists between isoenzymes and different peroxidases (Marklund, S. Ohlsson, P. I., Opara, A. and Paul, K. G. (1974) Biochim. Biophys. Acta 350, 304-313), which limits the generality of a given assay.
Hydroperoxides are key intermediates of prostaglandin, thomboxane, leukotriene, and lipoxin biosynthesis as well as initial products of lipid peroxidation (Pace-Asciak, C. R., Smith, W. L. (1983) in The Enzymes Vol. 16 (Boyer, P.D. ed.) Academic Press New York, pp. 544-603; Aust, S. D. and Svingen, B. A. (1982) in Free Radicals in Biology, Vol. V. (Pryor, W. A. ed.) Academic Press Orlando, pp. 1-28). An increasing body of evidence suggests that they attain significant levels in tissues and body fluids and that they are important regulators of enzyme activity, inflammation, thombosis, and metastasis (Warso, M. A. and Lands, W. E. M. (1984) Clin. Physiol. Biochem. 2, 70; Lands, W. E. M., Kulmacz, R. J. and Marshall, P. J. (1984) in Free Radicals in Biology, Vol. VI (Pryor, W. A. ed.) Academic Press Orlando, pp. 39-62). This suggests the importance of identifying enzymes that reduce peroxides and quantifying their capacity for hydroperoxide reduction.